PROJECT SUMMARY The long-term goal of this work is to elucidate the fundamental mechanism of exocytosis-endocytosis coupling at the central nerve terminals. Many types of synapses routinely transmit high-frequency action potentials through high-rate vesicle fusion at active zones. Fused synaptic vesicles and their associated proteins must be retrieved by endocytosis. In addition to regenerating new synaptic vesicles for future use, it is critical for balancing the surface area of the nerve terminals and maintaining intact ultrastructures. Despite decades of extensive research, the mechanism of endocytosis at chemical synapses is not fully addressed, particularly at physiological temperature. Strong evidence suggests that different modes of endocytosis take place in response to different synaptic activity, and endocytosis is a few orders of magnitude slower than vesicle fusion. However, recent morphological studies propose an ultrafast endocytosis that only occurs at a physiological temperature and replaces other forms of endocytosis. This is an attractive model because it efficiently minimizes the imbalance of surface area of nerve terminals during high-rate vesicle fusion. On the other hand, this model is built on the statistics of static images of fixed synapses, and sufficient functional data are required to test and characterize endocytosis at physiological temperature. The complete change of endocytosis pathways into a new, clathrin-independent endocytosis mode also raises many interesting new questions. Dynamin 1 is a large GTPase that is required for clathrin-mediate endocytosis at synapses, but its role in other forms of endocytosis such as bulk endocytosis and ultrafast endocytosis is less clear and controversial. In this proposal, we will address these questions by capacitance recordings from presynaptic terminals at physiological temperature. The time-resolve capacitance measurement (Cm) has high temporal resolution and sensitivity and thus is a suitable approach. First, we will characterize synaptic endocytosis by high time- resolution Cm at physiological temperature. We will use the calyx of Held, a fast glutamatergic central synapse in the auditory brainstem. We will overcome several technical limits during Cm recordings using new strategies and extract any fast endocytosis that may be present at physiological temperature. Different synaptic activities, including spontaneous single vesicle endocytosis, will be monitored. Secondly, we will use dynamin-1 conditional knockout mice as a valuable genetic model; its endocytosis properties at physiological temperate will be studied in response to various synaptic activities. This should provide significant insight into dynamin 1 function in vivo. This project will advance the field a step further and address several key questions recently raised by the rapid progress in this field. It will advance our knowledge on the kinetics and molecular mechanism of exocytosis-endocytosis coupling at central synapses under a condition similar to in vivo, and we expect a broad impact on cell biology of neurons and neuroscience.